USPTO Patents Application 10681049

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property
Organization

International Bureau

(43) International Publication Date
29 January 2004 (29.01.2004)

PCT

INI II III I II II II II II I II I II II III II III

(10) International Publication Number

WO 2004/009536 Al

(51) International Patent Classification 7 : C07C 259/06,

A61K 31/16, A6 IP 35/00

(21) International Application Number:

PCT7EP2003/007794

(22) International Filing Date: 17 July 2003 (17.07.2003)
(25) Filing Language: English

(26) Publication Language:

(30) Priority Data:

102 33 412.9

English

23 July 2002 (23.07.2002) DE

(71) Applicants: 4SC AG [DE/DE]; Am Klopferspitz 19a,
82152 Martinsried (DE). G2M CANCER DRUGS
AG [DE/DE]; Georg-Speyer-Haus, Paul-Ehrlich-Strasse
42-44, 60596 Frankfurt am Main (DE).

(72) Inventors: MAURER, Alexander, B.; Rainweg 5, 61352
Bad Homburg (DE). HOEVELMANN, Sascha; Meers-
burger Strasse 11, 60386 Frankfurt/Main (DE). MARTIN,
Elke; Eggensteiner Strasse 85, 76187 Karlsruhe (DE).
HENTSCH, Bernd; Moerser Strasse 45, 47239 Duisburg
(DE). GASSEN, Michael; Hiltensperger Str. 44, 80796
Munchen (DE). KRAUS, Juergen; Hadorfer Strasse
11a, 82319 Starnberg (DE). KRAUSS, Rolf; Rontgen-
str. 7, 82152 Planegg -Martinsried (DE). VINCEK,
Adam-Spencer; Rosegger Strasse 5, 81245 Munchen
(DE).

(74) Agent: STREHL, SCHUBEL-HOPF & PARTNER;

Maximilianstrasse 54, 80538 Munchen (DE).

(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, HR, HU, ID, IL, HN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, Nl, NO, NZ, OM, PG, PH, PL, Ff, RO, RU, SC,
SD, SE, SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA,
UG, UZ, VC, VN, YU, ZA, ZM, ZW.

(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).

Declaration under Rule 4.17:

— as to applicant's entitlement to apply for and be granted a
patent (Rule 4.17(H)) for all designations

Published:

— with international search report

— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

^ (54) Title: NOVEL COMPOUNDS AS HISTONE DEACETYLASE INHIBITORS

(57) Abstract: The present invention is directed to

\f) f Cri2 C\ compounds of the general formula (I) or pharmaceutical

^\ In I** / 1 \ acceptable salts or physiologically functional derivatives

( | ) thereof wherein: n is a non-aromatic ring system

containing two to seven carbon atoms, wherein the ring
N HOH system can contain one ore two double bonds; X is C, CH

orCH 2 ; Y is selected from C, CH, CH 2 , S, NR, CH 2 -CH 2 ,
H 2 C- -CH, HC- -CH 2 , C- -CH 2 , H 2 C- -C,or C - -C; one or
more of the hydrogen atoms can opionally be substituted by one or more substituents R ; each of the dotted lines means a single,
a double or triple bond with the exclusion of a combination of a triple with triple bond and a double with a triple bond; R is
J^. independently H, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy, -OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino,
^ halogene, haloalkyl, haloalkyloxy; R is H, an alkyl or cycloalkyl group; Z is CH, C, or P; p is 0 or I.

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Novel compounds as histone deacetylase inhibitors

The present invention relates to compounds as inhibitors of enzymes having histone
deacetylase activity, to the processes for the preparation of those compounds, and to
their use for the treatment of diseases which are associated with hypoacetylation of
histones and/or other molecules, or in which induction of hyperacetylation has a
beneficial effect for example by inhibition of proliferation and/or induction of
differentiation and/or induction of apoptosis in transformed cells, such as cancer.
Furthermore, the compounds are useful for the treatment of other proliferative
diseases, for therapy or prophylaxis of conditions associated with abnormal gene
expression.

BACKGROUND OF THE INVENTION

Local remodeling of chromatin is a key step in the transcriptional activation of genes.
Dynamic, changes in the nucleosomal packaging of DNA must occur to allow
transcriptional proteins to contact with the DNA template. One of the most important
mechanisms influencing chromatin remodeling and gene transcription are the
posttranslational modification of histones and other cellular proteins by acetylation
and subsequent changes in chromatin structure (Davie, 1998, Curr Opin Genet Dev
8, 173-8; Kouzarides, 1999, Curr Opin Genet Dev 9, 40-8; Strahl and Allis, 2000,
Nature 403, 41-4). In the case of histone hyperacetylation, changes in electrostatic
attraction for DNA and steric hindrance introduced by the hydrophobic acetyl group
leads to destabilisation of the interaction of histones with DNA. As a result,
acetylation of histones disrupts nucleosomes and allows the DNA to become
accessible to the transcriptional machinery. Removal of the acetyl groups allows the
histones to bind more tightly to DNA and to adjacent nucleosomes and thus to
maintain a transcriptionally repressed chromatin structure. Acetylation is mediated by
a series of enzymes with histone acetyltransferase (HAT) activity. Conversely, acetyl
groups are removed by specific histone deacetylase (HDAC) enzymes. Disruption of
these mechanisms gives rise to transcriptional misregulation and may lead to
tumorigenic transformation.

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Additionally, other molecules such as transcription factors alter their activity and
stability depending on their acetylation status. E.g. PML-RAR, the fusion protein
associated with acute promyelocyte leukemia (APL) inhibits p53 through mediating
deacetylation and degradation of p53, thus allowing APL blasts to evade p53
dependent cancer surveillance pathways. Expression of PML-RAR in hematopoietic
precursors results in repression of p53 mediated transcriptional activation, and
protection from p53-dependent apoptosis triggered by genotoxic stresses (X-rays,
oxidative stress). However, the function of p53 is reinstalled in the presence of HDAC
inhibitors implicating active recruitment of HDAC to p53 by PML-RAR as the
mechanism underlying p53 inhibition (Insinga et al. 2002, manuscript submitted).
Therefore, factor acetylation plays a crucial role in the anti-tumor activity of HDAC
inhibitors.

Nuclear hormone receptors are ligand-dependent transcription factors that control
development and homeostasis through both positive and negative control of gene
expression. Defects in these regulatory processes underlie the causes of many
diseases and play an important role in the development of cancer. Many nuclear
receptors, including T3R, RAR and PPAR, can interact with the corepressors N-CoR
and SMRT in the absence of ligand and thereby inhibit transcription. Furthermore, N-
CoR has also been reported to interact with antagonist-occupied progesterone and
estrogen receptors. N-CoR and SMRT have been shown to exist in large protein
complexes, which also contain mSin3 proteins and histone deacetylases (Pazin and
Kadonaga, 1997; Cell 89, 325-8). Thus, the ligand-induced switch of nuclear
receptors from repression to activation reflects the exchange of corepressor and
coactivator complexes with antagonistic enzymatic activities.

The N-CoR corepressor complex not only mediates repression by nuclear receptors,
but also interacts with additional transcription factors including Mad-1 , BCL-6 and
ETO. Many of these proteins play key roles in disorders of cell proliferation and
differentiation (Pazin and Kadonaga, 1997, Cell 89, 325-8; Huynh and Bardwell,
1998, Oncogene 17, 2473-84; Wang, J. et al., 1998, Proc Natl Acad Sci U S A 95,
10860-5). T3R for example was originally identified on the basis of its homology with
the viral oncogene v-erbA, which in contrast to the wild type receptor does not bind
ligand and functions as a constitutive repressor of transcription. Furthermore,

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mutations in RARs have been associated with a number of human cancers,
particularly acute promyelocytic leukemia (APL) and hepatocellular carcinoma. In
APL patients RAR fusion proteins resulting from chromosomal translocations involve
either the promyelocytic leukemia protein (PML) or the promyelocytic zinc finger
protein (PLZF). Although both fusion proteins can interact with components of the
corepressor complex, the addition of retinoic acid dismisses the corepressor complex
from PML-RAR, whereas PLZF-RAR interacts constitutively. These findings provide
an explanation why PML-RAR APL patients achieve complete remission following
retinoic acid treatment whereas PLZF-RAR APL patients respond very poorly
(Grignani et al., 1998, Nature 391, 815-8; Guidez et al., 1998, Blood 91, 2634-42; He
et al., 1998, Nat Genet 18, 126-35; Lin etal., 1998, Nature 391, 811-4). Furthermore,
a PML-RAR patient who had experienced multiple relapses after treatment with
retinoic acid has recently been treated with the HDAC inhibitor phenylbutyrate,
resulting in complete remission of the leukemia (Warrell et al., 1998, J. Natl. Cancer
Inst. 90,1621-1625).

By now, a clinical phase II trial with the closely related butyric acid derivative Pivanex
(Titan Pharmaceuticals) as a monotherapy has been completed demonstrating
activity in stage lll/IV non-small cell lung cancer (Keer et al., 2002, ASCO, Abstract
No. 1253). More HDAC inhibitors have been identified, with NVP-LAQ824 (Novartis)
and SAHA (Aton Pharma Inc.) being members of the structural class of hydroxamic
acids tested in phase I clinical trials (Marks et al., 2001, Nature Reviews Cancer 1,
194-202). Another class comprises cyclic tetrapeptides, such as depsipeptide
(FR901228 - Fujisawa) used successfully in a phase II trial for the treatment of T-cell
lymphomas (Piekarz et al., 2001, Blood 98, 2865-8). Furthermore, MS-27-275 (Mitsui
Pharmaceuticals), a compound related to the class of benzamides, is now being
tested in a phase I trial patients with hematological malignancies.

In Int. J. Chem. Kinet. 1997, 29, 729-735 3-Cyclopentyl-N-hydroxy-propionamide,
4-Cyclohexyl-N-hydroxy-butyramide and 2-Cyclohexyl-N-hydroxy-acetamide are
described (see also Berndt et al., 1992, Int. J. Chem. Kinet, 24, 695-701).

The crystal structure of a histone deacetylase like protein from the hyperthermophilic
bacterium aquifex aeolicus cocrystallized with the two inhibitors TSA and SAHA is
described by Finnin etal., 1999, Nature, 401, 188-193.

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Hydroxamic acids with at least one aromatic ring or ring system as histone
deacetylase inhibitors are described by Lavoie et al.,'2001, Bioorg. Med. Chem.
Letters 11, 2847-2850; Remiszewski et al., 2002, J. Med. Chem. 45, 4, 753-757;
Massa et al., 2001, J. Med. Chem. 44, 2069-2072; Sternson et al., 2001, Org. Lett.
3, 26, 4239-4242; Mai et al., 2002, J. Med. Chem. 45, 1778-1784; Woo et al., 2002,
J. Med. Chem. 45, 2877-2885.

In EP1 174438, WO0052033, WO01 18045, WO0118171, WO01 38322, WO01 70675,
WO9735990, W09911659, WO0226703, WO0230879 and WO0226696 hydroxamic
acids as histone deacetylase inhibitors are described.

In WO9805635 and WO9533709 hydroxamic acids as matrix metalloproteinase
inhibitors are described.

The compounds of the present invention are hydroxamic acids which are inhibitors of
enzymes having histone deacetylase activity. Due to their HDAC-inhibitory activity
they induce differentiation and/or apoptosis in a wide variety of cancer cells for three
reasons: (1) these enzymes are present in all cells and (2) pilot studies with model
compounds such as butyrate or TSA which are different from those described in this
invention had shown that HDAC inhibitors induce differentiation in a wide variety of
cells and (3) clinical efficacy has been demonstrated for several other HDAC
inhibitors unrelated to the presented compounds in the treatment of cancer patients.

The activity to induce differentiation and/or apoptosis in a wide variety of transformed
cells is a much more complex biological activity than only the inhibition of
proliferation. In the latter case it would not be evident, why only the proliferation of
transformed (tumor) but not of normal cells should be inhibited. The induction of
apoptosis, differentiation or more specifically re-differentiation in dedifferentiated
tumor cells provides a rationale why the compounds of this invention have beneficial
effects in a wide variety of tumors by induction of differentiation and/or apoptosis.

The histone deacetylase inhibitory activity of new compounds may be determined by
a number of state-of-the-art technologies such as transcriptional repression assay,
Western Blot analysis which detects acetylation of histone H3 and/or histone H4, or
by an enzymatic in vitro assay. Histone deacetylase inhibitors can be further

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characterized for their cytotoxic and growth inhibitory effects on tumor cell lines and
for their ability to modulate gene expression patterns in cells.

The present invention is directed to compounds of the general formula (I):

or pharmaceutical acceptable salts or physiologically functional derivatives
thereof wherein:

n is a non-aromatic ring system containing two to six carbon atoms, wherein
the ring system can contain one ore two double bonds;

X is C, CH or CH 2 ;

Y is selected from C, CH, CH 2 , S, NR, CH 2 -CH 2 ,

H 2 C- -CH HC- -CH 2j C" -CH 2j H 2 C- -C or O -C; one or more of
the hydrogen atoms can optionally be substituted by one or more
substituents FT;

each of the dotted lines means a single, a double or triple bond with the
exclusion of a combination of a triple with triple bond and a double with a triple
bond;

FT is independently H, -CN, alkyl, cycloalkyl, aminoalkyi, alkytamino, alkoxy,
-OH, -SH, alkylthio, hydroxyalkyl. hydroxyalkylamino, halogene, haloalkyl,
haloalkyloxy;

R is H, an alkyl or cycloalkyl group;
Z is CH, C, or P;

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p is 0 or 1 ; and

with the proviso that the following compounds are excluded:

An alkyl group, if not stated otherwise, is preferably a linear or branched chain
of 1 to 6 carbon atoms, preferably a methyl, ethyl, propyl, isopropyl, butyl,
f-butyl, isobutyl, pentyl or hexyl group, a methyl, ethyl, isopropyl or f-butyl group
being most preferred.

The term "alkyr, unless otherwise noted, is also meant to include those
derivatives of alkyl defined in more detail below as "unsaturated alkyl". An
unsaturated alkyl group is one having one or more double bonds or triple bonds,
preferably vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienylj, 2,4-
pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers.

The alkyl group in the compounds of formula (I) can optionally be substituted by
one or more substituents FT being as defined above.

An cycloalkyl group denotes a non-aromatic ring system containing 3 to 8
carbon atoms, wherein one or more of the carbon atoms in the ring can be
replaced by a group X, X being as defined above.

An alkoxy group denotes an O-alkyl group, the alkyl group being as defined
above.

An alkylthio group denotes an S-alkyl group, the alkyl group being as defined
above.

A hydroxyalkyl group denotes an HO-alkyl group, the alkyl group being as
defined above.

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An haloalkyl group denotes an alkyl group which is substituted by one to five
preferably three halogen atoms, the alkyl group being as defined above; a CF 3
being preferred.

An haloalkyloxy group denotes an alkoxy group which is substituted by one to
five preferably three halogen atoms, the alkoxy group being as defined above; a
OCF 3 being preferred.

A hydroxyalkylamino group denotes an (HO-alkyl) 2 -N-group or HO-alkyl-NH-
group, the alkyl group being as defined above.

An alkylamino group denotes an HN-alkyl or N-dialkyl group, the alkyl group
being as defined above.

An aminoalkyl group denotes an H 2 N-alkyl, monoalkylaminoalkyl. or
dialkylaminoalkyl group, the alkyl group being as defined above.

A halogen group is chlorine, bromine, fluorine or iodine, fluorine being preferred.

The invention also provides a pharmaceutical composition comprising a compound of
formula (I) in free form or in the form of pharmaceutical^ acceptable salts and
physiologically functional derivatives or prodrugs, together with a pharmaceutical^
acceptable diluent or carrier therefore.

The term "physiologically functional derivative" as used herein refers to compounds
like ethers, esters, N-alkylated or acetylated hydroxamic acids, 2,5-dihydro-[1 , 2, 4]-
oxodiazolyl or 4,5-dihydro-[1 , 2, 4]-oxodiazolyl, which are not pharmaceutical^ active
themselves but which are transformed into their pharmaceutical active form in vivo,
i.e. in the subject to which the compound is administered.

In another aspect, the present invention also provides a method for the treatment or
prophylaxis of a condition where there is an advantage in inhibiting histbne
deacetylase activity which comprises the administration of an effective amount of a

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compound of formula (I) and physiologically acceptable salts or physiologically
functional derivatives thereof.

The invention is also directed to the use of compounds of the formula (I) and of their
pharmacologically tolerable salts or physiologically functional derivatives for the
production of a medicament for the prevention and treatment of diseases, where
inhibition of histone deacetylase is of benefit

In addition, the present invention provides methods for preparing the desired
hydroxamic acides of the formula (I).

One method for the synthesis of compounds of the formula (I) comprises the
conversion of an acid (formula II) to the corresponding acid chloride (formula III) and
reacting the acid chloride with hydroxylamine (Watanabe et al., 1989, J. Org. Chem.,
54, 17, 4088-4097; Shishido et al., 1992, J. Org. Chem., 57, 10, 2876-2883).

formula (II)

formula (I)

formula (III)

Coupling reactions of acids of the formula (II) with hydroxylamine, other methods for
the preparation of compounds of the formula (I), are described by Woo et al., 2002, J.
Med. Chem. 45, 2877-2885; Knorr et al., 1989, Tetrahedron Lett., 30, 1927-1930,

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Carpino, 1993, J. Am. Chem. Soc, 115, 4397-4398 and Albericio etal., 1998, J. Org:
Chem., 63, 9678-9683.

Another method for the preparation of compounds of the formula (I) is the reaction of
the corresponding ester with hydroxylamine as described by Stowell et al., 1995, J.
Med. Chem., 38, 8, 1 41 1 -1 41 3.

In one preferred embodiments in the compounds of formula (I) the ring n including Z
can be cyclopentyl, cyclohexyl, cycloheptyl, cyclopent-1-enyl, cyclohex-1-enyl,
cyclohept-1-enyl, cyclopent-2-enyl, cyclohex-2-enyl, cyclohept-2-enyl, cyclohex-3-
enyl, cyclohept-3-enyl.

In another preferred embodiment in the compounds of the formula (I) of the present
invention the ring n including Z is cyclopentyl or cyclohexyl, and Y is selected from
CH, CH 2 , CH2-CH2, S, NR or p - 0, and Z is CH or P.

In another more preferred embodiment in the compounds of the formula (I) of the
present invention the ring n including Z is cyclopentyj or cyclohexyl, Y is selected
from CH, CH 2 , CH 2 -CH 2 , or p = 0 and Z is CH.

In another more preferred embodiment, none of the carbon atoms of the alkyl groups
is replaced by a group A.

Preferred compounds of the present invention are:
3-Cyclopentyl-N-hydroxy-propionamide;

3- Cyclohexyl-N-hydroxy-propionamide;

4- Cyclohexyl-N-hydroxy-butyramide;
2-Cycloheptyl-N-hydroxy-acetamide.

The compounds of the formula (I) according to the invention can form salts with
inorganic or organic acids or bases. Examples of such salts are, for example, alkali
metal salts, in particular sodium and potassium salts, or ammonium salts.

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The compounds of formula (I) may be obtained via various methods. In preferred
embodiments of the methods of the invention the two following methods of synthesis
are used.

Acids or acid chlorids of a-|3 unsaturated compounds of the formula (I) can be
obtained by hydrolysis of the corresponding ester to result the acid (Bojic et al., 1998,
354, 289-299) which can be converted with chlorinating agents (oxalylchloride,
thionylchloride, phosphorpenta chloride) into the acid chloride, a-p unsaturated
esters can be synthesized from the corresponding aldehyd by reacting them with
Carbethoxymethylene)triphenylphosphorane (PhP=CHCOOEt) (Maryanoff et aL,
1989, Chem. Rev. 89, 863-927).

Other methods for preparing different acids are described by Mancuso et ah, 1981,
Synthesis, 165-185; or Bal et aL, 1 981 , Tetrahedron, 37, 2091-2096.

The compounds of the present invention can be used for a variety of human and
animal diseases, preferably human diseases, where inhibition of histone deacetylase
activity is beneficial.

Therefore the compounds according to the invention and medicaments prepared
therewith are generally useful to induce the differentiation and/or apoptosis of cells
such as undifferentiated tumor cells. The therapeutic effect of the invention may arise
through one or more mechanisms, including but not limited to, the regulation of cell
proliferation, cell activation, cell survival, cell differentiation, cell cycle, cell maturation
and cell death or to induce systemic changes in metabolism such as changes in
sugar, lipid or protein metabolism, the inhibition of new blood vessel formation (anti-
angiogenesis), the inhibition of tumor spread into other organs (anti-metastatic), the
inhibition of tumor spread into neighboring normal structures (anti-invasive) or the
promotion of programmed cell death (apoptosis).

They can also be used to support cell generation poiesis, including blood cell growth
and generation (prohematopoietic effect) after depletion or destruction of cells, as
caused by, for example, toxic agents, radiation, immunotherapy, growth defects,
malnutrition, malabsorption, immune dysregulation, anemia and the like or to provide

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a therapeutic control of tissue generation and degradation, and therapeutic
modification of cell and tissue maintenance and blood cell homeostasis.

The compounds according to the invention and medicaments prepared therewith are
also useful for the treatment of a disease which is associated with gene-specific
hypoacetylation of histones or other molecules, such as p53. Additionally, the
compounds of the present invention may also be used in the treatment of conditions
where the suppression of anti-apoptotic genes, such as BCL-XL and other BCL
family members, or the induction of tumor suppressor activity of molecules such as
p21 and/or p53, is required.

The compounds according to the invention and medicaments prepared therewith are
also suitable for the treatment of diseases in which the induction of hyperacetylation
of histones has a beneficial effect resulting in differentiation and/or apoptosis of a
patient's tumor cells and thus causing a clinical improvement of the patient's
condition. Examples of such diseases include but are not limited to, skin cancer,
melanoma, estrogen receptor-dependent and independent breast cancer, ovarian
cancer, testosteron receptor-dependent and independent prostate cancer, renal
cancer, colon and colorectal cancer, pancreatic cancer, bladder cancer, esophageal
cancer, stomach cancer, genitourinary cancer, gastrointestinal cancer, uterine
cancer, astrocytomas, gliomas, basal cancer and squameous cell carcinoma,
sarcomas as Kaposi's sarcoma and osteosarcoma, head and neck cancer, small cell
and non-small cell lung carcinoma, leukemia, lymphomas and other blood cell
cancers, Keratoakantoma, Bowen Disease, cutaneous T-Cell Lymphoma and also for
the treatment of pre-malignant lesions (such as Actinic Keratose).
The combinatorial treatment of the present invention is particularly useful for treating
minimal residual tumor disease or tumor metastases.

Additionally, the invention may also be beneficial by reverting inappropriate gene
expression in diseases based on aberrant recruitment of histone deacetylase activity
such as thyroid resistance syndrome, or in other conditions associated with abnormal
gene expression, such as inflammatory disorders, diabetes, thalassemia, cirrhosis,
protozoal infection, or the like and all types of autoimmune diseases, in particular
rheumatoid arthritis, rheumatoid spondylitis, all forms of rheumatism, osteoarthritis,
gouty arthritis, multiple sclerosis, insulin dependent diabetes mellitus and non-insulin

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dependent diabetes, asthma, rhinitis, uveithis, lupus erythematoidis, ulcerative colitis;
Morbus Crohn, inflammatory bowel disease, as well as other chronic inflammations,
chronic diarrhea and of inflammations of the skin and/or mucosa (such as Psoriasis,
Ichtiosis, Acne). The invention also relates to the use for the protection from UV light
and for the treatment of sun burn.

Furthermore, the compounds according to the invention and medicaments prepared
therewith are useful for the treatment of other proliferative diseases such as
psoriasis, fibrosis, warts and other dermatological disorders. The terms "proliferative
disease", and "cell proliferation", are used interchangeably herein and relate to an
unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is
undesired, such as, neoplastic or. hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, pre-malignant and
malignant cellular proliferation, including malignant neoplasms and tumors, cancers,
leukemias, psoriasis, bone disease, fibroproliferative disorders (e.g. of connective
tissues), and atherosclerosis. Any type of cell may be treated, including but not
. limited to, lung, colon, breast, ovarian, prostate, liver, pancreas, brain, and skin and
any treatment of disorders involving T-cells such as aplastic anemia and DiGeorge
syndrome, Graves' disease.

The invention encompasses also compounds of formula (I) which are metabolized in
patients to a compound of the presented formula. The embodiments described in this
invention apply to such compounds as well.

The present invention also concerns a diagnostic method to identify tumors
comprising the step of testing in vitro whether a tumor is responsive to treatment
either with compounds of formula (I) or in combination with established tumor
therapeutics. The method preferably comprises the method for the identification of
genes regulated by these treatments. In a particular embodiment, the diagnostic
method comprises the use of nucleic acid technology, preferably of hybridization or
polymerase chain reaction for detection. Other types of nucleic acid technology,
however, may be employed. In another embodiment the method comprises the use
of specific antibodies against differentially regulated proteins for detection. For this
purpose proteins encoded by the genes showing deregulation of their expression

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upon combinatorial treatment using formulations of this invention and derivatives
thereof would be expressed e.g. in recombinant expression systems and antibodies
directed against these proteins would be generated. Subsequently such antibodies
could be used (or patterns of antibodies) to characterize the status of a tumor or
tumor cells for diagnostic and/or prognostic reasons.

In general the present invention provides novel possibilities to treat various human
diseases. Applicants found that the HDAC inhibitory and cellular differentiation-
inducing activity of compounds of formula (I) can be used successfully in combination
with well established and clinically used therapeutic drugs for the treatment of tumor
cells of different origins. Such compound based combinatorial treatment is
considered to generate superior therapeutic success in human tumor patients than
the corresponding therapeutic drugs used on their own. It is an object of the present
invention to provide combinatorial therapeutic approaches using the presented
compounds for the treatment of cancer. Such combinatorial treatments could result in
a decrease of the therapeutic doses of e.g. chemotherapeutic reagents required and
could thus limit the currently observed, partly very serious side effects of frequently
used therapies.

Aspects of the present invention are the combination of compounds of formula (I)
with, but not restricted to, therapeutic principles currently in clinical use or in clinical
development, such as

Chemotherapeutic or cytotoxic drugs (e.g. 5-FU, taxol, cisPlatinum,

camptothecin, gemcitabine, doxorubicine, irinothecan)

differentiation inducing drugs (e.g. vitamin D, retinoic acid, cytokines such as

11-3, 11-6, SCF, G-CSF, GM-CSF, TNF)

Radiation therapy (e.g. x-rays or gamma rays)

immunological approaches (antibody therapy, vaccination)

combined immunotherapeutic/cytotoxic approaches (e.g. antibodies

conjugated with cytotoxic components)

anti-angiogenesis approaches.

The compounds and salts thereof can be formulated, as pharmaceutical compositions
(e.g. liquids, suspensions, emulsions, lozenges, cachets, ampoules, suppositories,

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pessaries, ointments, gels, pastes, sprays, lotions, oils, boluses, electuaries,
aerosols, powders, granules, tablets, pills, capsules, injections, solutions, foams,
creams, enemas and the like) comprising at least one such compound alone or in
admixture with pharmaceutical^ acceptable carriers, excipients and/or diluents. The
pharmaceutical compositions can be formulated in accordance with a conventional
method.

Specific dose levels for any particular patient will be employed depending upon a
variety of factors including the age, body weight, general health, sex, diet, and prior
medication, and the severity of the particular disease of the patient, and the activity of
specific compounds employed, time of administration, route of administration, rate of
excretion, the duration of the treatment, other drugs, compounds, and/or materials
used in combination- It will be appreciated that the appropriate dosage of the active
compounds, and compositions comprising the active compounds, can vary from
patient to patient. Determining the optimal dosage will generally involve balancing of
the level of therapeutic benefit against any risk or deleterious side effects of the
treatments of the present invention.

Administration in vivo can be effected in one dose, continuously or intermittently
throughout the course of treatment. Methods of determining the most effective means
and dosages of administration are well known to those of skill in the art and will vary
with the formulation used for therapy, the purpose of the therapy, the target cell being
treated, and the subject being treated. Single or multiple administrations can be
carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 0.1 to
about 500 mg per kilogram body weight preferably 0.1 to 100 mg per kilogram body
weight of the subject per day. Where the active ingredient is a salt, an ester, prodrug,
or the like, the amount administered is calculated on the basis the parent compound
and so the actual weight to be used is increased proportionately.

Figures

Figure 1 shows the relief of HDAC-dependent transcriptional repression in a reporter
cell line, UAS TK-luc, after 24 hours treatment with Compound 1, 2 and 3. Depicted

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is the fold induction of luciferase expression after treatment of cells at the indicated
concentrations of Compounds 1 , 2 and 3.

Figure 2 shows the induction histone H4 hyperacetylation in 293T cells after
treatment with Compounds 1 , 2 and 3 for six hours at the indicated concentrations.

Figure 3 shows the inhibition of in-vitro HDAC activity in nuclear extracts from HeLa
cells after treatment with Compounds 1, 2 and 3 at the indicated concentrations.
Relative HDAC activity is shown as percent of activity of untreated nuclear extracts.

Figure 4 shows the inhibition of in-vitro HDAC activity of recombinant HDAC1 ,
HDAC6, and HDAC8 enzymes after treatment with Compound 3 at the indicated
concentrations. Relative HDAC activity is shown as percent of activity of untreated
HDAC enzymes.

Figure 5 shows on the protein level the down-regulation of the anti-apoptotic
molecule BCL-XL and up-regulation of the cell cycle inhibitor p21 in K562 cells
induced after treatment with Compounds 1, 2 and 3 for 36 hours at the indicated
concentrations.

Figure 6 shows the concentration of Compounds 1, 2 and 3 inducing a 50%
reduction in cellular biomass (IC 50 ) after treatment of various cancer cell lines for 72
hours.

Figure 7 shows the induction of apoptosis in BV-173 cells after treatment for 24
hours with Compounds 1, 2 and 3 at the indicated concentrations. Apoptosis is
demonstrated by the increase of a sub-G1 population in Propidium Iodine treated
cells, which is reduced to baseline levels after treatment with a pan-caspase inhibitor
Z-VAD.

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EXAMPLES
Example 1

Synthesis of Compound 1 (3-Cvclopentvl-/V-hvdroxv-propionamide) .

To a solution of 3-Cyclopentyl-propionyl chloride (11.0 mL, 11.6 g, 72.0 mmol) in
dichloromethane (50 mL) hydroxylamine hydrochloride (5.00 g, 72.0 mmol) and
sodium bicarbonate (12.0 g, 144 mmol) were added. After stirring for 24 h at room
temp, the reaction was quenched by addition of saturated ammonium chloride
solution (50 mL). The layers were separated. The water layer was extracted with
ethyl acetate (4 x 50 mL). The solvent of the combined organic layers was removed
in vacuo. Precipitation of the crude product out of ethyl acetate by adding petroleum
ether yielded the hydroxamate (5.25 g, 33.4 mmol, 46%) as a white solid. 1 H NMR
(300 MHz, [De-DMSO]: 5 = 0.74-0.91 (m, 2 H), 1.05-1.25 (m, 4 H), 1.36 (q, J = 7.4
Hz, 4 H), 1.54-1.71 (m, 5 H), 1.94 (t, J = 7.6 Hz, 2 H), 8.59 (s, 1 H) and 10.28 (s, 1 H)
(NH and OH). 13 C NMR (75 MHz, [De-DMSO]: 5 = 25.6, 26.0, 29.7, 32.4, 36.5, 169.2
(C(=0)NHOH). MS: m/z calcd for (C 8 H 14 N0 2 ) [M+H] + 158; found 158.

Example2:

Synthesis of Compound 2 (3-Cyclohexvl-A/-hvdroxv-propionamide):
To a solution of 3-Cyclohexyl-propionyl chloride (12.1 mL, 12.6 g, 72.0 mmol) in
dichloromethane (50 mL) hydroxylamine hydrochloride (5.00 g, 72.0 mmol) and
sodium bicarbonate (12.0 g, 144 mmol) were added. After stirring for 24 h at room
temperature the reaction was quenched by addition of saturated ammonium chloride
solution (50 mL). The layers were separated. The water layer was extracted with
ethyl acetate (4 x 50 mL). The solvent of the combined organic layers was removed
in vacuo. Precipitation of the crude product out of ethyl acetate by adding petroleum
ether yielded the hydroxamate (7.21 g, 42 mmol, 58%) as a white solid. 1 H-NMR (300
MHz, [De-DMSO]: 5 = 0.95-1 .1 2 (m, 2 H), 1 .38-1 .69 (m, 6 H), 1 .62-1 .77 (m, 3 H), 1 .94
(t, J = 7.6 Hz, 2 H), 8.58 (s, 1 H) and 10.28 (s, 1 H) (NH and OH); 13 C-NMR (75 MHz,
[De-DMSO]: 6 = 25.0, 31.8, 32.0, 32.3, 39.5, 169.5 (C(=)NHOH); MS: m/z calc. for
(CgHuNOij) [M+HT 172; found 172.

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Example 3:

Synthesis of Compound 3 (3-Cvclohexvl- N-hydroxv-acrvlamide)

3-Cyclohexyl-acrylic acid ethyl ester: to a cooled solution of oxalyl chloride (11.1 mL,
105 mmol) in dichloromethane (250 mL) was added at -78 °C a solution of
dimethylsulfoxide (14.6 mL, 206 mmol) in dichloromethane (250 mL). After 5 min at
the same temperature cyclohexylmethanol (10.8 mL, 87.5 mmol) and after additional
5 min triethylamine (60.7 mL, 438 mmol) were added. The reaction remained at -78
°C for two hours and then let warm to room temperature. The solvent was removed in
vacuo. The resulting aldehyde was used for the next step without further purification.
The crude cyclohexanecarbaldehyde was dissolved in toluene (200 mL) and ethanol
(150 mL). After 15 min of stirring at 70 °C carbethoxy-methylene
triphenylphosphorane (33.6 g, 96.5 mmol) was added in one portion. Stirring was
continued for additional 24 hours. The solvent was removed in vacuo. The product
was obtained by flash chromatography in 78 % yield (12.5 g). 1 H NMR (300 MHz,
[De-DMSO]: 8 = 1.07-1.34 (m, 8 H, CH 3 and cyclohexyl-CH 2 ), 1.60-1.80 (m, 5 H,
cyclohexyl-CHa), 2.04-2.18 (m, 1 H, cyclohexyl-CH); 4.16 (q, J = 7.1 Hz, 2 H, Et-
CH 2 ), 5.74 (dd, J = 15.8 Hz and J = 1.5 Hz, 1 H, C=C-H), 6.89 (dd, J = 15.8 Hz and J
= 6.8 Hz, 1 H, C=C-H).

3-Cyclohexyl-acrylic acid: to a solution of 3-Cyclohexyl-acrylic acid ethyl ester (7.00
g, 38.4 mmol) in dioxane (200 mL) was added lithium hydroxide (4.03 g, 96.1 mmol)
dissolved in water (70 mL). Because LiOH fell out methanol (170 mL) was added to
the reaction. After 4 hours at 70 °C 3/4 of the solvent was removed in vacuo. 2 N HCI
was added until the pH reached 3. The mixture was treated with ammonium chloride
(150 mL) saturated solution. The product was extracted with dichloromethane (200
mL x 3). The combined organic phases were washed with saturated sodium solution
(50 mL) and dried with magnesium sulfate. After filtration the solvent was removed in
vacuo. The liquid was left at room temperature for 48 hours for crystallization to
occur. The colourless crystalline product (2.01 g, 13.1 mmol) was obtained in 34%
yield. 1 H NMR (300 MHz, [D 6 -DMSO]: 6 = 1.01-1.37 (m, 5 H, cyclohexyl-CHg), 1.55-
1.79 (m, 5 H, cyclohexyl-CHa), 2.06-2.21 (m, 1 H, cyclohexyl-CH); 5.69 (dd, J = 15.7
Hz and J = 1.5 Hz, 1 H, C=C-H), 6.76 (dd, J - 15.7 Hz and J = 6.8 Hz, 1 H, C=C-
H).12.01 (broad s, 1 H, OH). 13 C NMR (75 MHz, [D 6 -DMSO]: 5 = 25.1, 25.4, 31.1

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(cyclohexyl-CHa), 39.4 (cyclohexyl-CH), 119.5 and 153.4 (C=C) 167.3 (COOH). MS:
m/z calcd for (CgHuOa) [M+HT 1 55; found 1 55.

3-Cyclohexyl-N-hydroxy-acrylamide (K1 00003283): to a solution of 3-Cyclohexyl-
acrylic acid (5.30 g, 31.3 mmol) in DMF (100 mL) was added EDC (1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (7.80 g, 40.7 mmol) and
HOBt (1-hydroxybenzotriazole hydrate) (5.07 g, 37.6 mmol). After stirring for 30 min
at room temperature hydroxylamine hydrochloride (3.26 g, 47.0 mmol) and
triethylamine (6.94 mL, 50.1 mmol) were added. After additional 18 hours stirring at
room temperature the precipitate was filtered and washed with DMF. The DMF
phases were combined. The solvent was concentrated in vacuo. 1 N HCI (50 mL)
was added. After extraction with ethyl acetate (3 x 100 mL) and dichloromethane (2 x
100 mL) the organic layers were combined, washed with 1 N HCI (10 mL) and
sodium chloride saturated solution (10 mL) and dried with magnesium sulfate. The
solvent was removed in vacuo. The product was crystallized from ethyl acetate by
addition of petroleum ether. This step removed large amounts of HOBt, the
substance was then further purified by preparative HPLC using an acetonitrile / water
gradient. This yielded (1.54 g, 9.08 mmol) of 3-Cyclohexyl-N-hydroxy-acrylamide. 1 H
NMR (300 MHz, [D 6 -DMSO]: 6 = 0.99-1.35 (m, 5 H) and 1.56-1.73 (m, 5 H)
(cyclohexyl-CH 2 ), 2.00-2.13 (m, 1 H, cyclohexyl CH), 5.67 (d, J = 15.7 Hz, 1 H, C=C-
H), 6.58 (dd, J = 15.7 and J = 6.3 Hz, 1 H, C=C-H), 8.77 (s, 1 H) and 10.49 (s, 1 H)
(NH and OH). 13 C NMR (75 MHz, [D 6 -DMSO]: 8 = 25.2, 25.5, 31.5 (cyclohexyl-CHa),
41.1 (cyclohexyl-CH), 118.8 and 147.1 (C=C), 162.8 (C(=0)NHOH). MS: m/z calcd
for (C9H 15 N02) [M+H] + 170; found 170.

Example 4

Compounds 1, 2 and 3 inhibit HDAC activity, induce histone H4 hyperacetylation,
and relieve transcriptional repression in a reporter cell line (Fig. 1 to 4). Histone
hyperacetylation correlates with an open, de-condensed chromatin structure and
gene activation, while hypoacetylation correlates with chromatin condensation and
transcriptional repression. Acetylation is mediated by a series of enzymes with
histone acetyltransferase (HAT) activity. Conversely, acetyl groups are removed by
specific histone deacetylase (HDAC) enzymes. Disruption of these mechanisms

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gives rise to transcriptional misregulation. Transcription factors such as thyroid
hormone receptor, PPAR5, retinoic acid receptor, N-CoR and AML/ETO recruit
HDAC enzymes and thereby repress transcription when bound to a specific promoter
region. This latter effect can be reenacted with a constitutive promoter containing
UAS elements which recruits fusion proteins containing the heterologous DNA-
binding domain of the yeast Gal4 protein fused to one of the above mentioned
transcription factors. In the absence of the Gal4-fusion protein the reporter gene has
a high basal transcriptional activity due to the presence of binding sites for other
transcription factors in the thymidine kinase promoter.

Methods

Transcriptional reporter gene assay. The transcriptional assay for repressor activity
exploits activation and derepression of a Gal4-dependent reporter gene (Hildebrand
et al., 2001, J Biol Chem 276, 9889-95 ; Maurer et aL, 2002, Blood 99, 2647-52).
This assay is performed with specifically constructed permanent cell lines. 293T cell
were stably transfected with a UAS TK luciferase reporter plasmid (Heinzel et al.,
1997 Nature 387, pp43-48) and an expression plasmid for the Gal4 DNA binding
domain fused to an HDAC-dependent repressor molecule. While Gal4 fusion proteins
repress this activity, HDAC inhibitors induce relief of this repression which can be
detected as an increase in reporter gene activity (e.g. by luciferase activity detection
assay).

Induction ofhistone hyperacetylation. These acetylated histones can be detected by
Western Blot analysis of whole cell extracts from histone deacetylase Inhibitor-
treated 293T cells using antibodies specific for the acetylated N-terminal lysine
residues of histones H4 (Gottlicher et al., 2001 , EMBO J 20, 6969-78).

In vitro inhibition of recombinant HDAC's. The determination of histone deacetylase
activity in HeLa nuclear extracts or recombinant HDAC proteins from High5 insect
cells is based on the specific deacetylation of an artificial substrate (Fluor de Lys,
Biomol). The substrate turn over may be detected and quantified by fluorometry. By
addition of a potential HDAC inhibitor the hydrolysis of the substrate is constrained

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resulting in a decreased fluorometric signal. IC 5 o values may be calculated from
dose-response curves.

The assay is separated in two steps: in the first step the substrate (Fluor de Lys /
Biomol KM 04) is hydrolysed by histone deacetylases. In step two HDAC activity is
terminated and the fluorophore is activated by the addition of a developer (Developer
/ Biomol KM 05). Nuclear extracts from HeLa cells (Biomol KM 40) or recombinant
proteins and the potential HDAC inhibitor are mixed with reaction buffer (Biomol Kl-
143) to a total volume of 25|il per well of a 96 well plate. 25pl substrate (1:100
dilution in reaction buffer) per well are added to start the reaction. A negative control
without histone deacetylase activity and a positive control without HDAC inhibitor are
treated likewise. The reaction is stopped after 15-60min. by adding 50|il developer
(1:20 dilution in reaction buffer). After another 15min. incubation time at room
temperature the fluorescence signal is stable for 60min and may be detected by a
fluorescence reader (excitation filter: 390nm, emission filter: 460nm).

Results

As can be seen in Figure 1, the Gal4 fusion protein represses the baseline activity of
the TK promoter and the subsequent luciferase expression. Addition of Compounds
1 , 2 and 3 relieve this repression as measured by increased expression and activity
of luciferase in lysates of treated cells after 24 hours. Both, Compound 1 and 2
induce luciferase reporter gene expression after stimulation of cells for 24 hours
starting at a concentration of 8 (2-3 fold) with a maximum induction of more than
20 fold at 200 |xM. Compound 3, however, induces expression of reporter gene
already at a concentration of 1 .6 juM with a maximum induction of almost 40 fold at
40 \M.

Since histone deacetylase inhibitors, shift the enzymatic balance between histone
acetyl transferases (HATs) and histone deacetylases (HDAC's) towards HATs by
blocking HDAC's, they induce the accumulation of N-terminally hyperacetylated
histones H4. This can be seen in Figure 2 by the induction of histone
hyperacetylation after treatment of 293T cells for 6 hours with Compounds 1 , 2 and
3. Hyperacetylation can be seen with Compounds 1 and 2 at minimum
concentrations of 1,6 jaM with a maximum induction. at 40 jxM, whereas compound 3
induces hyperacetylation starting at 320 nM.

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Since most HDAC's reside in the nucleus, highest HDAC activity can be measured in
nuclear extracts. As demonstrated in Figure 3, HDAC activity in nuclear extracts is
dramatically reduced in the presence of Compounds 1, 2 and 3, with a 50% reduction
of HDAC activity at a concentration of 14.7 \M for Compound 1, at 6.1 jxM for
Compound 2, and at 0.9 uM for Compound 3.

Results obtained with Compound 3 in nuclear extracts could be confirmed with
recombinant HDAC's. As demonstrated in Figure 4, HDAC activity of recombinant
HDAC1, HDAC6, and HDAC8 is dramatically reduced in the presence of Compound
1 and 2, with a 50% reduction of HDAC activity at a concentration of 0.3 to 0.4 uM for
Compound 3.

Example 5

Induction of growth arrest, apoptosis and down-regulation of BCL-X L after treatment
of cancer cells with Compounds 1, 2 and 3. The treatment of tumor cell lines with
histone deacetylase inhibitors leads to histone- hyperacetylation and the
transcriptional regulation of target genes. Although the discrete mechanism of action
varies, cancer therapy still depends on an ability to engender apoptosis in cancer
cells as a final common pathway. HDAC inhibitors have already been shown to
induce apoptosis in certain cancer cells through down-regulation of the anti-apoptotic
molecules, such as BCL-X L and BCL-2. Anti-apoptotic BCL2 family members seem to
be involved in resistance of tumors to apoptosis. For example, high expression of
BCL-Xl is found in many human cancers and is often a negative prognostic factor.
Accordingly, downregulation of BCL-X L expression in certain cancer cells either
induces apoptosis directly or sensitizes cells to apoptotic stimuli.
In general, induction of apoptosis can be exploited therapeutically using HDAC
inhibitors in cancer therapy

Methods

Protein expression profiling. The expression pattern induced by histone deacetylase
inhibitors can be monitored by Western Blot analysis with antibodies against p21 and
BCL-Xl using whole cell extracts of cells treated with the respective compounds at

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the indicated concentrations. Modulation of protein expression is exemplified by the
induction of expression of the cell cycle inhibitor/tumor suppressor p21 and the
suppression of expression of the anti-apoptotic molecule BCL-Xi_.

Growth inhibiton of tumor cell lines. The reduction in cellular biomass was measured
by SRB-assay. For this assay cells were seeded in 96 well culture dishes at densities
between 3000 and 8000 cells per well. After recovery of 24 hours cells were cultured
for 48-72 hours in the absence or presence of the indicated concentrations of
compounds. Synergistic reduction in total cellular biomass was assayed through
SRB-assay by adding the compounds in the medium at the indicated concentrations
and cultivate the cells for further 48 hours.

Cells were fixed with cold Trichloracetat (TCA) producing a final TCA concentration
of 10%. After 1 hour of incubation at 4°C the cells were washed five times with water
and air dried. Fixed cells were stained for 30 minutes with 0,4% (wt/vol)
Sulforhodamine B (SRB) dissolved in 1% acetic acid and washed four times with 1%
acetic acid to remove unbound dye. After air drying bound dye was solubilized with
10 mM unbuffered Tris base (pH 10,5) for 5 minutes. Optical densities (OD) were
read on a Molecular Devices Versa Max tunable microplate reader at 520-550 nm.
Four test wells for each dose-response were set in parallel with 12 control wells per
cell line. Measurement of the cell population density at time 0 (T 0 ; the time at which
the drug was added) was also made from 12 reference wells of cells fixed with TCA
just prior to drug addition to the test plates. Background OD of complete medium with
5% FBS fixed and stained as described above was also determined in 12 separate
wells. From the unprocessed OD data from each microtiter plate the background OD
measurements (i.e. OD of complete medium plus stain and OD of cells at T 0 ) were
subtracted thus giving the reduction of cellular biomass of the cells.

Measurement ofApoptosis. FACS analysis of the cell cycle by propidium iodide (PI)
staining. The cell cycle can be divided into four different sections: During G 0 /rphase
cells are in senescence or proliferate, in S-phase cells start replicating their DNA,
and during <32- and M-phase cells undergo mitosis. The cellular DNA content
correlates with cell cycle progression: while cells in G 0 /i phase possess one set of
chromosomes, cells in G2- and M-phase possess two full sets of chromosomes.
Cells in S-phase are still replicating their DNA and therefore exhibit a DNA content

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between one and two sets of chromosomes. Degradation of DNA is a marker for
apoptosis. The sub-G1 area indicates the hypodiploid DNA peak corresponding to
cells with fragmented DNA undergoing apoptosis Using a dye that intercalates with
DNA (such as propidium iodide - PI) the cellular DNA content was determined.
Between 5x1 0 5 - 1x10 6 cells were seeded out in cell culture dishes and treated for
the desired time with the desired amount of test compound. After the incubation cells
were harvested, washed in cold PBS, and the cell pellet was resuspended in 1ml
70% ethanol (-20°C). These cell pellets may be shelved for several month.
For PI staining 3ml cold sodium citrate solution (38mM, pH 7,4) was added, cells
were pelleted and stained with 500ul sodium citrate solution (38mM, pH 7,4)
containing 50ug/ml PI and 5ug/ml RNase. After a 30 minute incubation time at 37°C
in darkness, cells were analyzed via FACS analysis. The sub-G r peak constitutes the
amount of apoptotic cells.

Results

The expression of the growth arrest signaling tumor suppressor protein p21 (p21/waf)
and the anti-apoptotic protein BCL-X L was analyzed upon treatment with Compounds
1, 2 and 3. A down-regulation of the latter is regarded as one important pre-requisite
for the induction of apoptosis. Figure 5 shows that Compounds 1, 2 and 3 in fact
induces increased protein expression levels of p21 and at the same time down-
regulates BCL-Xl protein levels. Both, Compound 2 and 3 induce p21 expression and
BCL-XL downregulation at 8 \M, whereas Compound 1 induces p21 expression at
40 [M and BCL-XL downregulation at 200 [M.

Growth of various human tumor cell lines of breast, colon, pancreas and prostate
origin were inhibited by Compounds 1, 2, and 3 (Figure 6). The concentrations
inducing 50% growth arrest ranged from 8 to 70 \M (mean 33 uM) for Compound 1 ,
from 5 to 26 \M (mean 15 uM) for Compound 2, and from 0.5 to 8 \M (mean 2.6 jaM)
for Compound 3.

Studies using the BV-173 cell line consolidated the activity of Compound 1 and 2 as
inducers of programmed cell death (apoptosis). Examples of this type of analysis are
presented in Figure 7. The sub-G1 area (M1) indicates the hypodiploid DNA peak
corresponding- to cells with fragmented DNA undergoing apoptosis, which is

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dramatically increased to 50 - 60 % in cells treated with Compound 1 at 100 \xM,
Compound 2 at 50 \xM and Compound 3 at 5 jxM. This apoptotic process is however
completely blocked in the presence of a pan-caspase inhibitor, Z-VAD, which again
confirms the induction of apoptosis by Compounds 1 , 2, and 3.

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Claims

1 . Compounds of the general formula (I)

£CH 2

NHOH

or pharmaceutical acceptable salts or physiologically functional derivatives
thereof wherein:

n is a non-aromatic ring system containing two to seven carbon atoms,

wherein the ring system can contain one ore two double bonds;
X isC, CHorCH 2 ;

Y is selected from C, CH, CH 2 , S, NR, CH 2 -CH 2 ,

H 2 C--CH HC--CH 2 C--CH 2f H 2 C--C >or C--C; one or more of
the hydrogen atoms can optionally be substituted by one or more
substituents R x ;

each of the dotted lines means a single, a double or triple bond with the
exclusion of a combination of a triple with triple bond and a double with a triple
bond;

R' is independently H, -CN, alkyl, cycloalkyl, aminoalkyl, alkylamino, alkoxy,
-OH, -SH, alkylthio, hydroxyalkyl, hydroxyalkylamino, halogene, haloalkyl,
haloalkyloxy;

R is H, an alkyl or cycloalkyl group;
Z is CH, C, or P;
p is 0 or 1; and

with the proviso that the following compounds are excluded:

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PCT/EP2003/007794

2. The compound of claim 1 , wherein n = cyclopentyl or cyclohexyl.

3. The compound of claim 1 , wherein n = cyclopentyl or cyclohexyl and Z is CH.

4. A pharmaceutical composition comprising a compound as defined in any of
claims 1 to 3 in free form or in the form of pharmaceutical^ acceptable salts or
physiologically functional derivatives.

5. A compound according to any of claims 1 to 3, including the compounds
excluded in claim 1 for the use as a medicament.

6. The use of a compound according to claim 5 in the manufacture of a
medicament for use in treatment of a disease or a therapeutic indication as
inhibitors of enzyms having histone deacetylase activity.

7. The use of a compound according to claim 5 in the manufacture of a
medicament for use in treatment of a disease or a therapeutic indication in
which inhibition of histone deacetylase activity is beneficial.

8. The use according to claim 6 where the human histone deacetylase is
selected from the group consisting of HDACs 1-10 or a member of the SIR2
protein family.

9. The use of a compound according to claim 5 in the manufacture of a
medicament for the induction of differentiation of cells.

10. The use of a compound according to claim 5 in the manufacture of a
medicament for the induction of differentiation of transformed cells.

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PCT/EP2003/007794

11. The use of a compound according to claim 5 in the manufacture of a
medicament for the induction of apoptosis of transformed cells.

12. The use of a compound according to claim 5 in the manufacture of a
medicament for the inhibition of proliferation of transformed cells.

13. The use of a compound according to claim 1 for the inhibition of histone
deacetylase activity.

14. The use of a compound according to claim 5 in the manufacture of a
medicament, in which the induction of hyperacetylation of histones has a
beneficial effect.

15. The use of a compound according to claim 5 in the manufacture of a
medicament for use in treatment of a disease or a therapeutic indication
selected from the group consisting of skin cancer, melanoma, estrogen
receptor-dependent and independent breast cancer, ovarian cancer, prostate
cancer, renal cancer, colon and colorectal cancer, pancreatic cancer, head
and neck cancer, small cell and non-small cell lung carcinoma, leukemias and
other types of blood cell cancer and endocrine disease based on aberrant
recruitment of histone deacetylase such as thyroid resistance syndrome.

L

16. The use of a compound according to claim 5 in the manufacture of a
medicament for use in the inhibition of abnormal gene expression such as
inflammatory disorders, diabetes, thalassemia, cirrhosis or protozoal infection.

17. A process for the preparation of a compound according to claim 1 which
comprises the step of reacting an acid of formula (II)

formula (II)

(wherein n, X, Y, Z, and p are as defined in ciaim 1)

WO 2004/009536 PCT/EP2003/007794

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or an acid chloride of formula (III)

formula (III) .

(wherein n, X, Y, Z, and p are as defined in claim 1)
with hydroxylamine.

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IntematiorlW^ppllcatlon No

PCT/EP 03/07794

A. CLASSIFICATION OF SUBJECT MATTER .

IPC 7 C07C259/06 A61K31/16 A61P35/00

According to International Patent Classification (IPC) or to both national classification and (PC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 7 C07C

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

BEILSTEIN Data, EPO-Internal , CHEM ABS Data, PAJ, INSPEC, WPI Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category ° Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

KIRBY, GORDON W. ET AL: "Asymmetric

induction in the Di els-Alder reactions

of .alpha. -hydroxy acylnitroso compounds"

retrieved from STN

Database accession no. 120:134396

XP002264490

abstract

& JOURNAL OF THE CHEMICAL SOCIETY, PERKIN
TRANSACTIONS 1: ORGANIC AND BIO-ORGANIC.
CHEMISTRY (1972-1999) (1993), (13),
1397-402 ,

1-3

Further documents are listed In the continuation of box C.

Patent family members are listed in annex.

• Special categories of cited documents :

'A' document defining the general state of the art which is not

considered to be of particular relevance
•E' earlier document but published on or after the international

filing date

document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
•O' document referring to an oral disclosure, use, exhibition or
other means

"P* document published prior to the international filing date but
later than the priority date claimed

■T" later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

"X" document of particular relevance; the claimed Invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

"Y" document of particular relevance; the claimed invention
cannot be considered to Involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
In the art.

document member of the same patent family

Date of the actual completion of the international search

10 December 2003

Date of mailing of the international search report

29/12/2003

Name and mailing address of the ISA

European Patent Office, P.B. 5818 Patentlaan 2
NL-2280HVRfjswijk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Authorized officer

Bedel, C

Foim PCTASA/210 (second sheet) (July 1992)

page 1 of 4

INTERNATIONAL SEARCH REPORT

InternatlofflPVppllcatlon No

PCT/EP 03/07794

C.(Contlnuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category 0 Citation of document, with mdication.where appropriate, of the relevant passages

Relevant to claim No.

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

LION, C. ET AL: "New decontamination

reagents. Destruction of paraoxon and an

yperite-related compound by some

hydroxamic acids"

retrieved from STN

Database accession no. 115:183465

XP002264491

abstract

& BULLETIN DES SOCIETES CHIMIQUES BELGES
(1991), 100(8), 617-21 ,

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

KIRBY, GORDON W. ET AL: "Asymmetric

induction in the Di els-Alder reactions

of. alpha.- hydroxyacylnitroso compounds'

retrieved from STN

Database accession no. 111:115115

XP002264492

abstract

& TETRAHEDRON LETTERS (1988), 29(47),
6173-4 ,

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

GASPARINI, GIULIO M. : "The preparation and

properties of trial kylacetohydroxamic

acids: effect of the neoalkyl structure

with regard to the solubility, the

stability an some extractive capacities"

retrieved from STN

Database accession no. 92:93866

XP002264493

abstract

& GAZZETTA CHIMICA ITALIANA (1979),
109(6-7), 357-63 ,

1-3

1-3

1-3

Foim PCT/1SA/210 (continuation of second shoot) (July 1992)

page 2 of 4

INTERNATIONAL SEARCH REPORT

tntematioMRppllcatlon No

PCT/EP 03/07794

C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category ° Citation of document, with indication .where appropriate, of the relevant passages

Relevant to claim No.

DATABASE CA c 0nline!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

GASPARINI, GIULIO M. : "The

trial kylacetohydroxamic acids as selective

extractants in the reprocessing of

irradiated nuclear fuels"

retrieved from STN

Database accession no. 92:66434

XP002264494

abstract

& SPECIAL VOLUME - CANADIAN INSTITUTE OF
MINING AND METALLURGY (1979), 21(2 PROC.
INT. SOLVENT EXTR. CONF., 1977), 654-60 ,

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

LAPPA, M. ET AL: "Preparation and
properties of some trial kylacetohydroxamic
adds: stabilization of the hydroxamic
add function by the tertiary alkyl group
Part II"

retrieved from STN

Database accession no. 85:142583

XP002264495

abstract

& COM. NAZ. ENERG. NUCL., 'RAPP. TEC!
(1975), (RT/CHI(76)3), 42 PP. ,

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

WINTERNITZ, F. ET AL: "Reduction of
hydroxamic acids with double metal
hydrides"

retrieved from STN

Database accession no. 55:32651

XP002264496

abstract

& BULLETIN DE LA SOCIETE CHIMIQUE DE
FRANCE (1960) 509-14 ,

PATENT ABSTRACTS OF JAPAN

vol. 013, no. 536 (C-660),

29 November 1989 (1989-11-29)

& JP 01 221371 A (HOKKO CHEM IND CO LTD),

4 September 1989 (1989-09-04)

abstract

1-3

1-3

1-3

1-3

-/-

Form PCT/1SA/210 (continuation of second sheet) (July 1992)

page 3 of 4

INTERNATIONAL SEARCH REPORT

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PCT/EP 03/07794

C(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category" I Citation o( document, with indication.wUere approprlale, of the relevant passages

Relevant to claim No.

DATABASE CAPLUS 'Online!

CHEMICAL ABSTRACTS SERVICE , COLUMBUS,

OHIO, US;

INAMASU, SHUJI:

" . al pha. -Substituted- . beta . -Hydroxybutyroh

ydroxamic Acids"

XP002264497

Retrieved from STN

Database accession no : 84:135326

3ibs tr > 3tct

& JP 50 121218 A (JPN KOKAI TOKKYO K0H0)
23 September 1975 (1975-09-23)
page 135, right-hand column

US 3 994 997 A (SINGERMAN GARY M)
30 November 1976 (1976-11-30)
column 3, line 62 -column 4, line 25
column 4, line 41

DATABASE CA 'Online!

CHEMICAL ABSTRACTS SERVICE, COLUMBUS,

OHIO, US;

OYAMA, HIROSHI ET AL: "Preparation and

formulation of cyclic N-acyl hydroxy 1 amine

derivatives as agricultural and

horticultural fungicides"

retrieved from STN

Database accession no. 112:98566

XP002264498

8.L) s"t r* 8tc*t

& JP 01 221371 A (H0KKO CHEMICAL INDUSTRY

CO., LTD., JAPAN)

4 September 1989 (1989-09-04)

WO 01 70675 A (METHYLGENE INC)
27 September 2001 (2001-09-27)
page 114; example 54
page 208; example 54
claim 1

1-3

1-3,17

1-3

1-16

Form PCT7ISA/210 (continuation ot second shoet) (July 1992}

page 4 of 4

INTERNATIONAL SEARCH REPORT

IntematloraKpplIcatlon No

PCT/EP 03/07794

Patent document

Publication

Patent family

Publication

cited in search report

date

member(s)

date

JP 01221371

A

04-09-1989

NONE

JP 50121218

A

23-09-1975

NONE

US 3994997

A

30-11-1976

US

4059699 A

22-11-1977

JP 1221371

A

04-09-1989

NONE

WO 0170675

A

27-09-2001

AU

4870101 A

03-10-2001

CA

2404002 Al

27-09-2001

EP

1280764 A2

05-02-2003

WO

0170675 A2

27-09-2001

JP

2003528074 T

24-09-2003

US

2002115826 Al

22-08-2002

Form PCT/lSA/210 (paten! family amax) (July 1992)